Platinum (Pt) based therapies have proven to be curative treatments for a subset of cancers including the majority of testicular cancers. A large number of other cancer types including ovarian and lung, respond to Pt-based therapies which typically employ either cisplatin or carboplatin. Despite good initial responses in these cancers, tumor recurrence and resistance represent a significant and continuing clinical problem. The efficacy of these Pt-based therapies is a function of the formation of Pt-DNA adducts versus the removal of these adducts via DNA repair pathways. Removal of cisplatin-DNA lesions from the genome is catalyzed by the nucleotide excision repair (NER) pathway and is detrimental to treatment efficacy. In addition, while resistance to Pt-based therapies is typically multifactorial, clinical resistance often contains a DNA repair component. The goals of this work are to elucidate the molecular mechanism by which cisplatin-DNA damage is recognized and repaired by the NER pathway and to determine how perturbing the pathway influences cisplatin efficacy. Three Specific Aims are proposed to achieve the stated goals. In Aim 1 we will continue our study of the DNA damage recognition process by NER proteins. We will expand our focus to include the damage DNA binding protein (DDB) and the TFIIH complex. Building on the work accomplished in the previous grant periods with replication protein A (RPA), XPA and more recently XPC/hHR23B, we will use a novel combination of in vitro methodologies to construct a comprehensive structural, kinetic and biochemical model of the cisplatin-DNA recognition process by NER proteins. In Aim 2 we will employ a chemical genetics approach and develop small molecule inhibitors of NER DNA damage recognition proteins. Using these molecular tools we will determine how perturbing DNA damage recognition proteins influence in vitro DNA replication, repair and recombination pathways. In the third and final Aim we will assess how these inhibitors and perturbations of proteins involved in the damage recognition process influence cell proliferation, cell cycle progression, and ultimately cisplatin activity. The knowledge and molecular tools generated by this novel, innovative approach will likely impact the development of therapies targeting these pathways to overcome clinical resistance to cisplatin. The ultimate goal of this research is to translate the curative Pt-based therapies evident in certain cancers, to a wider array of cancers, including ovarian and lung.